Response to T cell-dependent antigens is characteristically associated with a progressive increase of antibody affinity for the eliciting determinants. This affinity maturation of the humoral response stems from a somatic process of hypermutation of V-genes in activated B cells and the selection of high-affinity antibody mutants for clonal expansion. These mechanisms result in the generation of antibody affinities that may be three logs greater than that of early serum and is thought to be crucial to ensure efficacious antibody responses to pathogens. Although affinity maturation is a hallmark of the thymus-dependent response, increases in antibody affinity are small or altogether absent in aged individuals responding to newly encountered antigens. The absence of affinity maturation may be a significant component of age-associated immune dysfunction with significant clinical sequelae. The experiments outlined in this proposal utilize a novel combination of histological and molecular genetic methods. Briefly, the B lymphocytes responding to challenge by NP conjugates are identified in sections of frozen splenic tissue through the use of fluorochrome-, enzynme-, or radiolabeled reagents. Positive cells may then be picked from the section by the use of a micromanipulator. Genomic DNA from these recovered cells may then be amplified using two rounds of PCR amplification cycles and nested 5' and 3' oligonucleotide primers. This technique permits the somatic genetics of an immune response to be followed directly and in situ; it will be employed to identify the failed component(s) of the immune response which prevents the aged individual from making high-affinity antibodies.